Electroporation device and electroporation method

Abstract

To provide an electroporation device and an electroporation method that can appropriately apply an electric field to a suspension and suppress a decrease in the introduction efficiency of a bioactive substance into a biological material. An electroporation device includes a flow path device used for electroporation, which introduces a bioactive substance into a biological material by applying an electric field by a pair of electrodes to a suspension containing the bioactive substance and the biological material. This flow path device comprises a flow path body and a tube connected to the flow path body. The flow path body has a main flow path extending in the vertical direction, a pair of electrodes for applying an electric field to a suspension flowing through the main flow path, a supply port for supplying the suspension to the main flow path, and a discharge port for discharging the suspension to which the electric field has been applied to the outside of the flow path device. The tube is connected to the discharge port, and at least a part of the tube faces upward in the vertical direction.

Description

Electroporation device and electroporation method

[0001] The present invention relates to an electroporation device and an electroporation method for introducing a bioactive substance into a biologically derived substance by applying an electric field to a suspension containing the biologically derived substance and a bioactive substance, and in particular to an electroporation device and an electroporation method in which at least a portion of a tube attached to an outlet for discharging the suspension to which an electric field has been applied to the outside faces vertically upward.

[0002] In recent years, the number of approved adeno-associated virus (AAV) gene therapies has increased, necessitating reduced manufacturing costs and a stable supply of viral vectors. The triple transfection (TT) method is the most commonly used chemical transfection method for producing recombinant AAV (rAAV) gene therapy products. Meanwhile, physical methods for transfecting cells with various substances, such as electroporation, are also known (see Patent Documents 1 and 2). Hereinafter, electroporation may be abbreviated as EP. Electroporation involves the application of high-voltage electric pulses to cells, creating transient pores in the cell membrane through which plasmid DNA diffuses into the cells. However, approaches using standard static cuvettes and other existing approaches have low throughput and are not suitable for large-scale manufacturing.

[0003] Patent Documents 1 and 2 also disclose a continuous liquid feed type electroporation in which a suspension containing a biologically derived substance and a bioactive substance is passed through a flow path in which an electrode pair is installed.

[0004] Japanese Patent Application Laid-Open No. 2007-7430 U.S. Patent No. 1,1225,638

[0005] The present inventors are currently developing a unique continuous production platform and flow path device for electroporation. To scale up production, the inventors attempted to enlarge the flow path of the flow path device. This resulted in a decrease in the efficiency of introducing bioactive substances into biologically derived materials, which was not an issue with conventional microscale flow path devices. The present invention aims to provide an EP device and an EP method that can properly apply an electric field to a suspension and suppress a decrease in the efficiency of introducing bioactive substances into biologically derived materials.

[0006] The above-mentioned object can be achieved by the following configuration: Invention [1] is an EP apparatus including a flow path device used for EP, which applies an electric field to a suspension containing a biologically derived substance and a bioactive substance using an electrode pair to introduce the bioactive substance into the biologically derived substance, wherein the flow path device includes a flow path main body and a tube connected to the flow path main body, the flow path main body has a main flow path extending in the vertical direction, an electrode pair that applies an electric field to the suspension flowing in the main flow path, a supply port that supplies the suspension to the main flow path, and a discharge port that discharges the suspension to which the electric field has been applied to the outside of the flow path device, and the tube is connected to the discharge port, and at least a portion of the tube faces vertically upward.

[0007] Invention [2] is the EP device according to Invention [1], in which liquid is pumped from below to above in the vertical direction through the main flow path. Invention [3] is the EP device according to Invention [1] or [2], in which at least a portion of the tube extending from the outlet and adjacent thereto faces upward in the vertical direction. Invention [4] is the EP device according to any one of Inventions [1] to [3], in which a portion of the tube extending 3 cm or more from the outlet faces upward in the vertical direction from the outlet.

[0008] Invention [5] is the EP device according to any one of Inventions [1] to [4], in which the connection portion of the tube at the outlet of the exhaust port faces upward in the vertical direction. Invention [6] is the EP device according to any one of Inventions [1] to [5], in which the tube is located at a position extending 5 cm horizontally from the outlet of the exhaust port and at a position 2 cm or more above in the vertical direction. Invention [7] is the EP device according to any one of Inventions [1] to [6], in which the EP device has a discharge flow path that communicates with the main flow path and the exhaust port, at least a portion of the discharge flow path extends vertically, and the outlet of the exhaust port faces upward in the vertical direction. Invention [8] is an EP method comprising the steps of: mixing a culture solution containing a biologically derived substance obtained by culturing with a bioactive substance to obtain a suspension; and using the EP device described in any one of Inventions [1] to [6], transporting the suspension from below to above in the vertical direction of a main flow path, and applying an electric field to the suspension using an electrode pair to introduce the bioactive substance into the biologically derived substance.

[0009] According to the present invention, it is possible to provide an EP device that can properly apply an electric field to a suspension and suppress a decrease in the efficiency of introducing a bioactive substance into a biologically derived substance.

[0010] Fig. 1 is a schematic plan view showing a first example of an electroporation apparatus according to an embodiment of the present invention; Fig. 2 is a schematic cross-sectional view showing the first example of an electroporation apparatus according to an embodiment of the present invention; Fig. 3 is a schematic cross-sectional view showing an example of tube arrangement in the first example of an electroporation apparatus according to an embodiment of the present invention; Fig. 4 is a schematic cross-sectional view showing a second example of an electroporation apparatus according to an embodiment of the present invention; Fig. 5 is a schematic cross-sectional view showing an example of tube arrangement in the second example of an electroporation apparatus according to an embodiment of the present invention; Fig. 6 is a schematic cross-sectional view showing a third example of an electroporation apparatus according to an embodiment of the present invention.

[0011] The electroporation apparatus and electroporation method of the present invention will be described in detail below based on the preferred embodiments shown in the accompanying drawings. Note that the drawings described below are illustrative for explaining the present invention, and are simplified for the purpose of explaining the present invention, and the present invention is not limited to the drawings shown below. In the following, the symbol "to" indicating a range of values ​​includes the values ​​written on both sides. For example, when ε is a value ε α ~Number ε β That is, the range of ε is the number ε α and the number ε β The range includes ε α ≦ε≦ε β Furthermore, unless otherwise specified, the range of error generally accepted in the relevant technical field is included for specific angles, parallelism, perpendicularity, etc. Furthermore, unless otherwise specified, the range of error generally accepted in the relevant technical field is included for length, width, thickness, etc. Furthermore, unless otherwise specified, the range of error generally accepted in the relevant technical field is included for temperature, flow rate, electrical conductivity, electric conductivity, volume fraction, etc.

[0012] (First Example of Electroporation Apparatus) FIG. 1 is a schematic plan view showing a first example of an electroporation apparatus (EP apparatus) according to an embodiment of the present invention. FIG. 2 is a schematic cross-sectional view showing the first example of an electroporation apparatus according to an embodiment of the present invention. FIG. 2 is a cross-sectional view taken along line A-A in FIG. 1. Also, FIG. 1 does not illustrate the tube 13, piping 21, pumps 22a and 22b, culture device 23, power supply unit 24, control unit 25, tank 26, and mixer 30 shown in FIG. 2. The EP apparatus 10 shown in FIGS. 1 and 2 includes a flow path device 11 used for electroporation, which applies an electric field to a suspension Q containing a biologically derived substance and a bioactive substance using an electrode pair 17, thereby introducing the bioactive substance into the biologically derived substance. The suspension Q is continuously pumped between the electrode pair 17, and an electric field is applied to the suspension Q using the electrode pair 17, thereby introducing the bioactive substance into the biologically derived substance. The EP device 10 is a continuous liquid feed type electroporation device that performs EP by continuously feeding the suspension Q between the electrode pair 17. Performing EP by continuously feeding the suspension Q between the electrode pair 17 is also called continuous electroporation.

[0013] As shown in Fig. 2, the EP device 10 has a flow path device 11, pumps 22a and 22b, a power supply unit 24, and a control unit 25. Furthermore, for example, a culture device 23, which is a culture solution supply unit, is connected to the pump 22a of the EP device 10. The pump 22a is provided between the mixer 30 and the culture device 23. The culture device 23 does not constitute the EP device 10. Furthermore, a tank 26 and a mixer 30, which will be described later, do not constitute the EP device 10 either. Although Fig. 2 shows the culture device 23 as the culture solution supply unit, it may also be a tank that stores the suspension taken out from the culture device 23.

[0014] The flow path device 11 includes a flow path main body 12 and a tube 13 connected to the flow path main body 12. The flow path main body 12 has a main flow path 14 extending in the vertical direction z, an electrode pair 17 that applies an electric field to the suspension Q flowing through the main flow path 14, a supply port 15 that supplies the suspension Q to the main flow path 14, and a discharge port 16 that discharges the suspension Q to which the electric field has been applied to the outside of the flow path device 11. The tube 13 is connected to the discharge port 16 and is connected to the flow path main body 12. The tube 13 is made of a flexible material such as a silicone tube, for example.

[0015] In the EP device 10, the main flow path 14 of the flow path body 12 of the flow path device 11 is a linear flow path. Ｌ The cross-sectional shape of the main flow path 14 in a direction perpendicular to the horizontal plane Hp is, for example, a rectangle with all interior angles of 90°. The above-mentioned cross-sectional shape of the main flow path 14 is not limited to a rectangle with all interior angles of 90°. A supply port 15 for supplying the suspension Q to the main flow path 14 is provided on the front surface 12a of the flow path body 12 at one end 14a of the main flow path 14. A discharge port 16 for discharging the suspension Q to the outside is provided on the back surface 12b of the flow path body 12 at the other end 14b of the main flow path 14. The main flow path 14 and the discharge port 16 are connected by a discharge flow path 70. For example, the discharge flow path 70 is a linear flow path that extends in a direction parallel to the horizontal plane Hp. An opening of the discharge flow path 70 on the opposite side to the end 14b of the main flow path 14 is the discharge port 16, and an opening of the discharge flow path 70 on the opposite side to the end 14b of the main flow path 14 is the outlet 16a of the discharge port 16.

[0016] The flow path body 12 shown in FIG. 2 is, for example, in the extending direction D of the linear main flow path 14 with respect to the horizontal plane Hp. Ｌ The main flow path 14 extends in the vertical direction z. The extension direction D of the main flow path 14 Ｌ is a vertical direction z perpendicular to the horizontal plane Hp, and is at an angle of 90° with respect to the horizontal plane Hp, but the vertical direction z of the main flow path 14 is allowed to deviate by ±10° from 90°. ＬThe angle is 90°, with a tolerance of ±10° from 90°. The suspension Q is injected from the supply port 15, flows through the main flow path 14 along the direction Df shown in Figures 1 and 2, and is discharged from the discharge port 16. The direction Df shown in Figures 1 and 2 is also the direction from upstream to downstream of the main flow path 14, and is the direction from below to above in the vertical direction z. In the main flow path 14, the supply port 15 side is the upstream side, and the discharge port 16 side is the downstream side.

[0017] The width direction of the main flow channel 14 is parallel to the direction x perpendicular to the vertical direction z, and the extension direction D of the main flow channel 14 Ｌ The extending direction D of the main flow path 14 is also perpendicular to the Ｌ is the direction Df in which the suspension Q flows within the main flow path 14. In the EP device 10, the liquid transfer direction Dd of the suspension Q between the electrode pair 17 is a direction from below to above in the vertical direction z. The suspension Q flows within the main flow path 14 in a direction from below to above in the vertical direction z. In the above embodiment, the supply port 15 is provided on the surface 12a of the flow path main body 12 (see FIG. 2 ), and the discharge port 16 is provided on the back surface 12b of the flow path main body 12 (see FIG. 2 ). However, the present invention is not limited to this, and the supply port 15 and the discharge port 16 may each be provided on the surface 12a of the flow path main body 12 or on the back surface 12b of the flow path main body 12.

[0018] A pipe 21 is connected to the supply port 15. A mixer 30 is provided on the pipe 21. The pump 22a of the EP device 10 is connected to, for example, a culture device 23 via the pipe 21. The pump 22a pumps the suspension Q from below to above in the vertical direction z through the main flow path 14, and the suspension Q is continuously pumped between the electrode pair 17 in a direction from below to above in the vertical direction z. While the culture device 23 is shown in FIG. 2 as the culture solution supply unit, a tank for storing the culture solution removed from the culture device 23 may also be used. The culture device 23 contains a culture solution (not shown) containing a biologically derived substance and a culture medium. In the culture device 23, for example, cell culture is performed, and a culture solution containing cells and a culture medium is obtained. A tank 26 is connected to the mixer 30. A pump 22b is provided between the mixer 30 and the tank 26. A bioactive substance, for example, dispersed in liquid, is stored in the tank 26. The configuration of the tank 26 is not particularly limited as long as it can store a bioactive substance.

[0019] The mixer 30 mixes the culture solution and the bioactive substance. The mixture of the culture solution and the bioactive substance is called suspension Q, and suspension Q includes a culture medium. By using pump 22a to aspirate the culture solution from the culture device 23 and pump 22b to aspirate the bioactive substance from the tank 26, the culture solution from the culture device 23 and the bioactive substance from the tank 26 are mixed in the mixer 30 to produce suspension Q without replacing the culture medium with an electroporation (EP) buffer. The configuration of the mixer 30 is not particularly limited as long as it can mix the culture solution and the bioactive substance, and any known mixer can be used as appropriate. The suspension Q is supplied to the main flow channel 14 via the pipe 21 and the supply port 15 by the pumps 22a and 22b. In this way, the suspension Q is supplied to the main flow channel 14 without replacing the culture medium with an EP buffer. The suspension Q is transported between the electrode pair 17 in a vertical direction z, from below to above.

[0020] The configuration of the piping 21 and the pumps 22a, 22b is not particularly limited, and those known in the art can be used as appropriate depending on the viscosity of the suspension Q, the amount of suspension Q to be fed, etc. The piping 21 may be flexible, such as a silicone tube. The culture device 23 is not necessarily required. Instead of the culture device 23, for example, a tank (not shown) in which the suspension Q is stored may be used. In this case, the tank is connected to the pump 22a.

[0021] The electrode pair 17 that applies an electric field to the suspension Q has a first electrode 18 and a second electrode 19. The first electrode 18 has an electrode surface 18a, which is flat. The second electrode 19 has an electrode surface 19a, which is flat. The electrode surface 18a of the first electrode 18 and the electrode surface 19a of the second electrode 19 are arranged opposite to each other and parallel to each other. The electrode surface 18a of the first electrode 18 and the electrode surface 19a of the second electrode 19 are each arranged facing the inner surface 14c of the main flow channel 14. The electrode surface 18a of the first electrode 18 and the electrode surface 19a of the second electrode 19 each constitute the inner surface 14c of the main flow channel 14 and are in contact with the suspension Q. A space 20 between the opposing electrode surface 18a of the first electrode 18 and the electrode surface 19a of the second electrode 19 is the space between the electrode pair 17.

[0022] The first electrode 18 and the second electrode 19 of the electrode pair 17 are electrically connected to a power supply unit 24. A voltage is applied to the electrode pair 17 by the power supply unit 24. That is, the power supply unit 24 applies a voltage to the opposing first electrode 18 and second electrode 19. The configuration of the power supply unit 24 is not particularly limited as long as it can apply a pulse voltage to the electrode pair 17 and adjust the pulse width and pulse period. For example, a pulse power supply is used as the power supply unit 24.

[0023] The control unit 25 of the EP device 10 is connected to the pumps 22a and 22b and the power supply unit 24. The control unit 25 controls the operation of the pumps 22a and 22b. The control unit 25 also adjusts the magnitude, pulse width, and pulse period of the voltage applied by the power supply unit 24 to the electrode pair 17. The control unit 25 also adjusts the timing of the operation of the pump 22 and the timing of the application of voltage to the electrode pair 17 by the power supply unit 24. The control unit 25 may be configured, for example, by a computer that functions by executing a program, or may be a dedicated device configured with a dedicated circuit. The control unit 25 may be configured separately from the flow path device 11. The control unit 25 may be remotely controlled by the control unit 25. The configuration for remote control by the control unit 25 is not particularly limited and may be a known configuration. The above-mentioned computer and dedicated device each have, for example, a processor. The processor may be configured by one or more pieces of hardware, and the type of hardware is not limited. For example, the processor may be configured by hardware such as a CPU (Central Processing Unit), an MPU (Micro Processing Unit), a programmable logic device such as an FPGA (Field Programmable Gate Array), a dedicated circuit for executing specific processing such as an ASIC (Application Specific Integrated Circuit), a GPU (Graphic Processing Unit), or an NPU (Neural Processing Unit).

[0024] The flow path body 12 of the flow path device 11 shown in FIG. 2 is configured by, for example, stacking a first substrate 27, a flow path substrate 28, and a second substrate 29 in this order. The flow path substrate 28 has an opening 28a that becomes the main flow path 14. The first substrate 27 has a supply port 15 that communicates with the opening 28a of the flow path substrate 28. The second substrate 29 has a discharge flow path 70 and a discharge port 16 that communicate with the opening 28a of the flow path substrate 28. The supply port 15 penetrates the first substrate 27 in the stacking direction Ds of the first substrate 27, the flow path substrate 28, and the second substrate 29. The discharge port 16 penetrates the second substrate 29 in the stacking direction Ds. As described above, the flow path substrate 28 is sandwiched between the first substrate 27 and the second substrate 29, and therefore the main flow path 14 is formed by the opening 28a of the flow path substrate 28. Note that the second substrate 29 may have the supply port 15 and the discharge port 16 instead of the first substrate 27, or the first substrate 27 may have one of the supply port 15 and the discharge port 16, and the second substrate 29 may have the other of the supply port 15 and the discharge port 16. Note that the stacking direction Ds is parallel to the direction y that is perpendicular to the vertical direction z. The direction y is parallel to the horizontal plane Hp.

[0025] A first electrode 18 is fitted into the first substrate 27. A second electrode 19 is fitted into the second substrate 29. The electrode surface 18a of the first electrode 18 faces the electrode surface 19a of the second electrode 19. As described above, the electrode surface 18a of the first electrode 18 and the electrode surface 19a of the second electrode 19 each constitute the inner surface 14c of the main flow channel 14. For example, when performing continuous liquid transfer EP, the electrode surface 18a and the electrode surface 19a of the second electrode 19 come into contact with the suspension Q when the suspension Q is supplied to the main flow channel 14. In the flow channel body 12 of the flow channel device 11 shown in FIG. 2 , the first substrate 27, the flow channel substrate 28, and the second substrate 29 may be fixed to each other by any method, including forming through-holes for assembly in each plate and fixing them with screws or adhesive. O-rings are preferably provided between the first substrate 27 and the flow channel substrate 28 and between the second substrate 29 and the flow channel substrate 28 as sealing members (not shown) to prevent leakage of the suspension Q.

[0026] In the EP device 10, the pump 22 delivers the suspension Q between the electrode pair 17, i.e., in the space 20, such that the delivery direction Dd of the suspension Q is from bottom to top in the vertical direction z. When an electric field is applied to the suspension Q using the power supply 24 to perform EP, and bubbles are generated between the opposing first and second electrodes 18 and 19, the generated bubbles have a lower specific gravity than the suspension Q and are therefore more likely to move in the vertical direction z than the suspension Q. Therefore, bubbles generated between the electrode pair 17 are removed from between the electrode pair 17 by moving in the vertical direction z without remaining between the electrode pair 17 due to the delivery of the suspension Q, thereby suppressing the adverse effects of the application of an electric field to the suspension Q. This allows the EP device 10 to properly apply an electric field to the suspension Q, thereby optimizing the introduction efficiency and utilization efficiency of the bioactive substance.

[0027] As described above, the tube 13 is connected to the outlet 16. At least a portion of the tube 13 faces upward in the vertical direction z from the outlet 16 toward the liquid feed direction. This makes it difficult for bubbles generated between the electrode pair 17 and removed from between the electrode pair 17 to be trapped by the outlet 16. This facilitates the bubbles to be discharged from the flow path main body 12 to the tube 13 along with the flow of the fed suspension Q, and the bubbles are discharged to the outside of the flow path main body 12. This allows the electric field to be applied appropriately to the suspension Q, and prevents a decrease in the efficiency of introducing the bioactive substance into the biologically derived material. The tube 13 only needs to face upward from the horizontal, based on the height of the outlet 16; in FIG. 2 , it faces diagonally upward. If the tube is oriented downward, for example, and bubbles are trapped in the outlet 16 and not discharged to the outside of the flow path main body 12, the bubbles may accumulate and enter the main flow path 14. In this case, air bubbles entering the main flow path 14 disrupt the flow velocity distribution of the suspension Q, reducing the efficiency of introducing the bioactive substance into the biologically derived material. In the EP device 10, the above-described arrangement of the tube 13 makes it difficult for air bubbles to be trapped at the outlet 16, thereby preventing a decrease in the efficiency of introducing the bioactive substance into the biologically derived material. In the EP device 10, the suspension Q is fed so that the feed direction Dd of the suspension Q is from below to above in the vertical direction z. This reduces the adverse effects of air bubbles between the electrode pair, but there is still room for improvement in the EP efficiency. This configuration is particularly effective in cases where air bubbles are likely to be generated, as described below. Furthermore, if trapped air bubbles break, the resulting pressure fluctuations can damage the biologically derived material after EP processing, potentially reducing the quality of the biologically derived material or the effectiveness of the introduced bioactive substance. In the EP device 10, the above-described arrangement of the tube 13 makes it difficult for air bubbles to be trapped at the outlet 16, thereby suppressing the above-described significant damage to the biological material. Furthermore, when there are many trapped air bubbles, if the trapped air bubbles coalesce and break, the pressure fluctuation caused by the bubble breakage becomes large.This causes significant damage to the biological material after EP processing, so the bubbles that are generated between the electrode pair 17 and removed from between the electrode pair 17 must not only be removed from between the electrode pair 17, but also must be trapped in the outlet 16, etc., so as not to remain within the flow path main body 12.

[0028] The quality of the biological product refers to, for example, cell viability. The effect of the introduced bioactive substance refers to, for example, the gene expression efficiency when a gene is introduced as a biological product into a cell as a biologically derived product. The efficiency of introducing a bioactive substance into the biological product is also referred to as electroporation efficiency. Furthermore, "tube 13 facing upward in the vertical direction z" means that, when a horizontal plane is set below tube 13 at the connection between outlet 16 and tube 13, the distance between the circumferential surface of tube 13 and the horizontal plane gradually increases from outlet 16 in a direction parallel to the horizontal plane.

[0029] As described above, bubbles generated between the electrode pair 17 and removed from between the electrode pair 17 are less likely to be trapped at the outlet 16, thereby suppressing a decrease in electroporation efficiency. Therefore, it is preferable that at least the vicinity 13a of the tube 13 extending from the outlet 16 faces vertically upward. The vicinity 13a of the tube 13 is within 3 cm from the outlet 16a of the outlet 16 of the tube 13. It is preferable that a portion of the tube 13 extending 3 cm or more from the outlet 16 faces upward in the vertical direction z from the outlet 16. This is more preferably 5 cm or more, and even more preferably 10 cm or more. The upper limit of the vicinity is 20 cm.

[0030] As shown in FIG. 2, the tube 13 is positioned at a position P １ At the position P 2 cm or more above the vertical direction z ２ 1. As a result, the tube 13 is positioned at a position P ２Since the position P faces upward in the vertical direction z, bubbles generated between the electrode pair 17 and removed from between the electrode pair 17 are more easily discharged from the outlet 16a of the outlet 16, and bubbles are less likely to be trapped in the outlet 16, which more reliably prevents a decrease in electroporation efficiency. １ is the horizontal distance W from the outlet 16a of the discharge port 16 １ is 5 cm. ２ is the position P １ The point P is on a line Lv that passes through the point P and extends in the vertical direction z. １ and position P ２ The distance W in the vertical direction z ２ The horizontal direction is the direction parallel to the horizontal plane Hp. １ and distance W ２ can be measured with a ruler or calipers.

[0031] FIG. 3 is a schematic diagram showing an example of tube arrangement in a first example of an electroporation apparatus according to an embodiment of the present invention. In FIG. 3, components identical to those in the flow channel device 11 shown in FIG. 2 are designated by the same reference numerals, and detailed description thereof will be omitted. As described above, at least a portion of the tube 13 faces upward in the vertical direction z. In this case, as shown in FIG. 3, it is preferable to have a holding unit 34 that holds the tube 13 at a predetermined position. The holding unit 34 includes, for example, a holding unit 34a that holds the tube 13 and a support unit 34b that maintains the holding unit 34a at a desired height in the vertical direction. It is preferable that the holding unit 34a hold the tube 13 without deforming the inner wall of the tube 13 and creating a constriction. It is preferable that the holding unit 34a be stopped at any position of the support unit 34b in the vertical direction z. This allows the placement position of the tube 13 to be adjusted. On the opposite side of the flow path body 12 from the holding portion 34 (highest position), the tube 13 may face downward in the vertical direction z. The tip of the tube 13 is connected to, for example, a container (not shown) that stores the suspension after EP.

[0032] 3, a connecting portion 32 is provided at the outlet 16a of the discharge port 16. The connecting portion 32 includes a cylindrical base 32c and a truncated cone-shaped tapered portion 32d connected to the base 32c from the outlet 16a side of the discharge port 16. The connecting portion 32 includes a linear passage 32a that passes through the base 32c and the tapered portion 32d in the longitudinal direction. The passage 32a is connected to the outlet 16a of the discharge port 16. The outer periphery of the tapered portion 32d expands outward from the opening surface 32b of the connecting portion 32 toward the outlet 16a of the discharge port 16. The connecting portion of the tapered portion 32d with the base 32c extends outward beyond the base 32c, thereby preventing the tube 13 from coming loose. The tube 13 is connected to the connecting portion 32 by covering the tapered portion 32d and the base 32c of the connecting portion 32. The connection portion 32 of the outlet 16a of the discharge port 16 faces upward in the vertical direction z. The direction of the connection portion 32 refers to the longitudinal direction of the passage 32a. This makes it difficult for air bubbles generated between the electrode pair 17 and removed from between the electrode pair 17 to be trapped at the discharge port 16. This makes it easier for air bubbles to be carried by the flow of the pumped suspension Q and discharged from the flow path main body 12 to the tube 13, making it easier for the air bubbles to be discharged to the outside of the flow path main body 12. Note that as long as at least a portion of the tube 13 faces upward in the vertical direction z as described above, the connection portion 32 may face horizontally. The connection portion 32 is a member for connecting the tube 13 to the flow path main body 12 and is made of, for example, hard plastic. The hard plastic is, for example, polycarbonate.

[0033] Furthermore, as shown in FIG. 2 , the flow path main body 12 preferably has an angle of greater than 90° between the opening surface 16b including the outlet 16a of the outlet 16 and the horizontal plane. This causes the opening surface 16b of the outlet 16 to face upward in the vertical direction z, making it difficult for bubbles generated between the electrode pair 17 and removed from between the electrode pair 17 to be trapped by the outlet 16 and quickly move to the tube 13, thereby suppressing the bubbles from being trapped in the outlet 16. The upper limit of the angle between the opening surface 16b of the outlet 16 and the horizontal plane is 180°, and in this case, the opening surface 16b and the horizontal plane are parallel. The opening surface 16b of the outlet 16 shown in FIG. 2 has an angle of greater than 90° with the horizontal plane and does not face upward. 3 , when the connecting portion 32 is provided at the outlet 16a of the discharge port 16, the opening surface 32b of the connecting portion 32 corresponds to the opening surface 16b including the outlet 16a of the discharge port 16. In any of the above-described configurations, as long as at least a portion of the tube 13 faces upward in the vertical direction z as described above, the tube 13 may be configured to have a horizontally extending portion (not shown) extending horizontally from the outlet 16a of the discharge port 16. The length of the horizontally extending portion of the tube 13 is, for example, preferably within 10 cm, more preferably within 5 cm, and even more preferably within 3 cm from the outlet 16a of the discharge port 16 of the tube 13.

[0034] (First Example of Electroporation Method) The first example of the electroporation method (EP method) is a method in which a suspension Q containing a biologically derived substance and a bioactive substance is continuously fed between the electrode pair 17, i.e., into the above-mentioned space 20, and an electric field is applied to the suspension Q by the electrode pair 17, thereby introducing the bioactive substance into the biologically derived substance. The first example of the EP method is a continuous liquid-feed EP method, and uses, for example, the EP device 10, culture device 23, tank 26, and mixer 30 shown in Figures 1 and 2. In the first example of the EP method, a step is performed in which the culture solution in the culture device 23 is aspirated by the pump 22a of the EP device 10, and the bioactive substance in the tank 26 is aspirated by the pump 22b, and the culture solution in the culture device 23 is mixed with the bioactive substance in the tank 26 in the mixer 30, without replacing the medium of the culture solution with an EP buffer. The pump 22a supplies the suspension Q to the main flow path 14 through the pipe 21 and the supply port 15, and the suspension Q is continuously pumped vertically from bottom to top between the electrode pair 17. At this time, a pulse voltage, for example, is applied to the electrode pair 17 by the power supply unit 24, and an electric field is applied to the suspension Q by the electrode pair 17, thereby carrying out a step of introducing a bioactive substance into the biologically derived material. In this manner, the EP method is carried out.

[0035] In the first example of the EP method, the flow direction Dd of the suspension Q between the electrode pair 17, i.e., in the space 20, is set to a direction from bottom to top in the vertical direction z. When EP is performed by applying an electric field to the suspension Q using the power supply unit 24 and bubbles are generated between the opposing first and second electrodes 18 and 19, the generated bubbles have a smaller specific gravity than the suspension Q and are therefore more likely to move in the vertical direction z than the suspension Q. Therefore, bubbles generated between the electrode pair 17 are removed from between the electrode pair 17 by moving in the vertical direction z without remaining between the electrode pair 17 due to the flow of the suspension Q, thereby suppressing the adverse effects of the application of an electric field to the suspension Q. This allows the first example of the EP method to properly apply an electric field to the suspension Q, thereby optimizing the introduction efficiency and utilization efficiency of the bioactive substance. Furthermore, even if the composition of suspension Q is such that bubbles are likely to be generated when an electric field is applied to suspension Q, if bubbles are generated between electrode pair 17 as described above, the generated bubbles have a smaller specific gravity than suspension Q and therefore move more easily in the vertical direction z than suspension Q. As suspension Q is fed, the bubbles do not remain between electrode pair 17 but move in the vertical direction z and are removed from between electrode pair 17, thereby suppressing the adverse effects of the application of an electric field to suspension Q. Furthermore, because at least a portion of tube 13 faces upward in the vertical direction as described above, bubbles generated between electrode pair 17 and removed from between electrode pair 17 are less likely to be trapped at outlet 16. Instead, the bubbles are more likely to be discharged from flow path main body 12 to tube 13 along with the flow of fed suspension Q, and are thus discharged to the outside of flow path main body 12. This allows for proper application of an electric field to suspension Q and suppresses a decrease in electroporation efficiency.

[0036] In a first example of the EP method, prior to EP, a culture step may be performed in which a culture solution containing a biologically derived material and a medium is obtained by cell culture, and a mixing step may be performed in which the culture solution is mixed with a bioactive substance to form a suspension. Typically, prior to EP, biologically derived material (e.g., cells) is removed from the culture solution and suspended in an EP buffer to obtain a suspension. Replacing this medium with an EP buffer is simply referred to as medium exchange or buffer exchange. In the first example of the EP method, the culture medium obtained in the culture step is mixed with a bioactive substance without being exchanged for an EP buffer, i.e., without adding an EP buffer, and the suspension is continuously pumped between the electrode pair 17. In the first example of the EP method, since the culture medium is not exchanged for an EP buffer, the resulting suspension contains more medium and has higher electrical conductivity than a suspension typically subjected to EP. Therefore, a large number of bubbles are generated when an electric field is applied. Even for such a suspension, the suspension is subjected to electroporation while moving vertically from bottom to top between the pair of electrodes, so that any bubbles generated between the pair of electrodes are removed from between the pair of electrodes by moving vertically, thereby suppressing the adverse effects of the application of an electric field to the suspension due to the bubbles. The culturing step is carried out in the culturing device 23 as described above. The mixing step is carried out in the mixer 30 by using the pumps 22a and 22b to suck the culture solution from the culturing device 23 and the bioactive substance from the tank 26 as described above.

[0037] (Second Example of Electroporation Apparatus) FIG. 4 is a schematic cross-sectional view showing a second example of an electroporation apparatus (EP apparatus) according to an embodiment of the present invention. FIG. 5 is a schematic cross-sectional view showing an example of the tube arrangement of the second example of the electroporation apparatus according to an embodiment of the present invention. In FIGS. 4 and 5, the same components as those in the EP apparatus 10 shown in FIGS. 1 and 2 are designated by the same reference numerals, and detailed description thereof will be omitted. The EP apparatus 10a differs from the EP apparatus 10 shown in FIGS. 1 and 2 in that it uses a sheath fluid s in addition to the suspension Q. Otherwise, it has the same configuration as the EP apparatus 10 shown in FIGS. 1 and 2. Like the EP apparatus 10 (see FIG. 2), the EP apparatus 10a also has a flow path device 50 including a flow path main body 12c and a tube 13, and the tube 13 is connected to the flow path main body 12c. Furthermore, for example, a pump 22a is connected to the culture apparatus 23. The pump 22a is provided between the mixer 30 and the culture apparatus 23. A tank 26 is connected to the mixer 30. A pump 22b is provided between the mixer 30 and the tank 26. The culture device 23, the tank 26, and the mixer 30 do not constitute the EP device 10a. The EP device 10a is a continuous liquid transfer type EP device that uses a suspension Q and a sheath liquid s, and performs EP by transferring the suspension Q and the sheath liquid s so that the suspension Q is sandwiched between the sheath liquid s, continuously transferring the suspension Q between an electrode pair 44, and applying an electric field to the suspension Q by the electrode pair 44. In the EP device 10a, the suspension Q is transferred so that the sheath liquid s sandwiches the suspension Q, and there are two sheath liquid flows.

[0038] In the EP apparatus 10a, the flow path body 12c of the flow path device 50 shown in Fig. 4 has a main flow path 40, a supply port 15 for supplying the suspension Q to the main flow path 40, a discharge port 16 for discharging the processed suspension that has undergone the continuous liquid transfer type EP, and a first supply port 41a and a second supply port 41b for supplying the sheath liquid s to the main flow path 40. The main flow path 40 is a linear flow path. The main flow path 40 extends in the extension direction D ＬThe cross-sectional shape of the main flow path 40 in the direction perpendicular to the horizontal plane Hp is, for example, a rectangle with all of the interior angles at 90°. The cross-sectional shape of the main flow path 40 is not limited to a rectangle with all of the interior angles at 90°. The flow path body 12c is, for example, a rectangle with all of the interior angles at 90° relative to the horizontal plane Hp. Ｌ The main flow path 40 extends in the vertical direction z. The extension direction D of the main flow path 40 Ｌ is a vertical direction z perpendicular to the horizontal plane Hp and has an angle of 90° with respect to the horizontal plane Hp, but the vertical direction z of the main flow path 40 is allowed to deviate by ±10° from 90°. In the main flow path 40 of the flow path body 12c, the supply port 15 side is the upstream side, and the discharge port 16 side is the downstream side.

[0039] As shown in FIG. 4 , a supply port 15 for supplying the suspension Q to the main flow channel 40 is provided at one end 40a of the main flow channel 40, for example, on the front surface 12a of the flow channel body 12c. A discharge port 16 for discharging liquids such as the suspension Q and the sheath fluid s to the outside is provided at the other end 40b of the main flow channel 40, for example, on the back surface 12b of the flow channel body 12c. In the main flow channel 40, the supply port 15 side is the upstream side, and the discharge port 16 side is the downstream side. The main flow channel 40 and the discharge port 16 are connected by a discharge flow channel 70. For example, the discharge flow channel 70 is a linear flow channel extending in a direction parallel to the horizontal plane Hp. The opening of the discharge flow channel 70 on the opposite side to the end 40b of the main flow channel 40 is the discharge port 16, and the opening of the discharge flow channel 70 on the opposite side to the end 40b is the outlet 16a of the discharge port 16. 4, a first supply port 41a for supplying the sheath fluid s to the main flow channel 40 is provided, for example, on the front surface 12a of the flow channel body 12c. A second supply port 41b for supplying the sheath fluid s to the main flow channel 40 is provided, for example, on the back surface 12b of the flow channel body 12c. For example, the first supply port 41a and the second supply port 41b are arranged in the extension direction D. Ｌ The positions in are the same.

[0040] A pipe 21 is connected to the supply port 15. A pump 22a is connected to the supply port 15 via the pipe 21 connected to the supply port 15. The pipe 21 is used to supply the suspension Q when the sheath fluid s is sent to the main flow path 40 so as to sandwich the suspension Q therebetween.

[0041] A pipe 21a is connected to the first supply port 41a. A pump 22c is connected to the first supply port 41a via the pipe 21a connected to the first supply port 41a. A tank 36 is connected to the pump 22c via the pipe 21c. A pipe 21b is connected to the second supply port 41b. A pump 22d is connected to the second supply port 41b via the pipe 21b connected to the second supply port 41b. The tank 36 is connected to the pump 22d via the pipe 21d. There are two pipes 21a and 21b because there are the first supply port 41a and the second supply port 41b for supplying the sheath fluid s. The two pipes 21a and 21b are used to supply the sheath fluid s when sending the sheath fluid s to the main flow path 40 so as to sandwich the suspension Q between them. The configurations of the pipes 21 a, 21 b and the pumps 22 c, 22 d are not particularly limited, and any known configurations in the art may be used as appropriate depending on the viscosity of the sheath fluid s, the amount of sheath fluid s to be delivered, etc. The pipes 21 a, 21 b may be flexible, such as silicone tubes.

[0042] Although the configuration includes pumps 22c and 22d, this is not limiting. A single pump may be provided for both the first supply port 41a and the second supply port 41b. That is, the sheath fluid s may be supplied to the flow path body 12c by a single pump. The tank 36 stores the sheath fluid s. The tank 36 is not particularly limited in its configuration as long as it can store the sheath fluid s. As described above, the supply port 15 is provided on the front surface 12a of the flow path body 12c, and the discharge port 16 is provided on the back surface 12b of the flow path body 12c. However, this is not limiting. For example, one of the supply port 15 and the discharge port 16 may be provided on the front surface 12a of the flow path body 12c, and the other on the back surface 12b. Furthermore, the locations of the first supply port 41a and the second supply port 41b are not particularly limited. They may be provided on the front surface 12a or the back surface 12b of the flow path body 12c, respectively.

[0043] An electrode pair 44 is provided in the main flow channel 40 downstream of a confluence 59 where the suspension flow and the sheath liquid flow confluence. The electrode pair 44 has electrodes arranged opposite to each other and applies an electric field to the suspension Q flowing through the main flow channel 40. The electrode pair 44 that applies the electric field to the suspension Q has a first electrode 45 and a second electrode 46 arranged opposite to each other. The first electrode 45 has an electrode surface 45a, which is flat. The second electrode 46 has an electrode surface 46a, which is flat. The electrode surface 45a of the first electrode 45 and the electrode surface 46a of the second electrode 46 are arranged opposite to each other and parallel to each other.

[0044] In the EP device 10a, the flow path main body 12c is basically configured to include, for example, a first substrate 52, a second substrate 54, a first inlet 56, a second inlet 58, and a flow path substrate 55 provided between the first substrate 52 and the second substrate 54. The first inlet 56, the first substrate 52, the flow path substrate 55, the second substrate 54, and the second inlet 58 are stacked in this order. A first electrode 45 is incorporated into the first substrate 52, and a second electrode 46 is incorporated into the second substrate 54, thereby constituting an electrode pair 44. The electrode surface 45a of the first electrode 45 and the electrode surface 46a of the second electrode 46 each constitute the inner surface 40c of the main flow path 40, and come into contact with the sheath fluid s when the suspension Q and the sheath fluid s are supplied to the main flow path 40 during EP, for example. A space 47 between the opposing electrode surface 45 a of the first electrode 45 and the opposing electrode surface 46 a of the second electrode 46 is between the electrode pair 44 .

[0045] As shown in Figure 4, the main flow path 40 is formed in a substantially straight line between the first substrate 52 and the second substrate 54. The main flow path 40 is provided in the flow path substrate 55. In the flow path main body 12c, the suspension also flows from the supply port 15 to the discharge port 16, and the liquid transfer direction Dd of the suspension Q between the electrode pair 44 is a direction from below to above in the vertical direction z. The second substrate 54 is provided with a discharge flow path 70 that communicates with the other end 40b of the main flow path 40 in the flow path substrate 55, and with a discharge port 16. The discharge port 16 protrudes, for example, from the opposite side of the second substrate 54 from the flow path substrate 55, i.e., protrudes from the back surface 12b of the flow path main body 12c.

[0046] The first introduction section 56 is provided with a through hole 56b extending in the stacking direction Ds of the first introduction section 56, first substrate 52, flow path substrate 55, second substrate 54, and second introduction section 58. Furthermore, the first substrate 52 is provided with a through hole 52b that communicates with the through hole 56b of the first introduction section 56 and extends in the stacking direction Ds. The through hole 52b communicates with the most upstream portion of the main flow path 40 via a suspension inlet 55d. The opening of the through hole 56b of the first introduction section 56 on the opposite side of the first substrate 52 is a supply port 15. The supply port 15 communicates with the most upstream portion of the main flow path 40 via the through hole 56b of the first introduction section 56 and the through hole 52b of the first substrate 52.

[0047] Furthermore, the first substrate 52 is provided with a through-hole 52a communicating with the main flow path 40. The second substrate 54 is provided with a through-hole 54a communicating with the main flow path 40. The through-holes 52a and 54a are provided at an angle with respect to the main flow path 40. The first introduction section 56 is provided with a through-hole 56a communicating with the through-hole 52a of the first substrate 52. Furthermore, the second introduction section 58 is provided with a through-hole 58a communicating with the through-hole 54a of the second substrate 54. The through-hole 56a communicates with the first supply port 41a and the through-hole 52a of the first substrate 52, extends from the first supply port 41a in the stacking direction Ds, and is inclined at the boundary with the first substrate 52 in the extension direction Ds of the main flow path 40. Ｌ The through-hole 56a having such a shape is conveniently referred to as a substantially L-shaped through-hole 56a. The through-hole 58a communicates with the second supply port 41b and the through-hole 54a of the second substrate 54, extends from the second supply port 41b in the stacking direction Ds, and intersects with the extension direction Ds of the main flow path 40 at the boundary with the second substrate 54. ＬThe through-hole 58a is bent in a direction perpendicular to the axis of the sheath fluid s. For convenience, the through-hole 58a having this shape will be referred to as a substantially L-shaped through-hole 58a. The through-hole 56a of the first introduction section 56 and the through-hole 52a of the first substrate 52, and the through-hole 58a of the second introduction section 58 and the through-hole 54a of the second substrate 54 are arranged symmetrically with respect to the main channel 40 extending in the vertical direction z. A sheath fluid flow path 64 extends from the first supply port 41a to the through-hole 56a and the through-hole 52a. A sheath fluid flow path 65 extends from the second supply port 41b to the through-hole 58a and the through-hole 54a. The sheath fluid flow paths 64 and 65 are flow paths through which only the sheath fluid s flows and which allow the sheath fluid s to flow into the main channel 40. The through-hole 52a communicates with a sheath fluid junction port 52c provided in the main channel 40, which will be described later. The through-hole 54a communicates with a sheath liquid confluence port 54c provided in the main channel 40, which will be described later.

[0048] The first supply port 41a communicates with the main channel 40 via a substantially L-shaped through-hole 56a provided in the first introduction section 56 and a through-hole 52a provided in the first substrate 52. The sheath fluid flow path 64 is provided so as to be inclined downstream with respect to the flow direction of the liquid in the main channel 40, and opens so that the sheath fluid S flows downstream of the suspension supply port and upstream of the electrode pair 44. The region on the upstream side of the main channel 40 through which only the suspension Q flows is the suspension flow path. The sheath fluid flow paths 64, 65 preferably have a region in which the flow path widens in the width direction toward the downstream side. As a result, even if the width of the main channel 40 is wide, the flow velocity distribution downstream is uniform, enabling uniform EP for the suspension Q. In this case, it is preferable that the suspension flow path also has a region in which the flow path widens in the width direction toward the downstream side, thereby achieving a uniform flow velocity distribution in the suspension flow path. The through-hole 56a (part of the sheath fluid flow path) may widen in the width direction, for example, downstream from the bent position of the approximately L-shaped flow path, so that the sheath fluid flow is uniform in the width direction. This also applies to the through-hole 58a (part of the sheath fluid flow path) described below. That is, the sheath fluid flow path 65 preferably has a region in the through-hole 58a where the flow path widens in the width direction downstream. As a result, even if the main flow path 40 is wide, the flow velocity distribution downstream becomes uniform, enabling uniform electroporation of the suspension Q. In this case, it is also preferable that the suspension flow path has a region where the flow path widens in the width direction downstream, so that the flow velocity distribution in the suspension flow path becomes uniform. Furthermore, the through-hole 56a may be provided across the first introduction part 56 and the first substrate 52.

[0049] On the other hand, the second supply port 41b communicates with the main channel 40 via a substantially L-shaped through hole 58a provided in the second introduction section 58 and a through hole 54a provided in the second substrate 54. Here, similar to the through holes 56a and 52a described above, the through holes 58a and 54a form the sheath fluid flow path 65. Therefore, the above description of the through holes 56a and 52a also applies to the through holes 58a and 54a. Note that the through hole 58a may be provided across the second introduction section 58 and the second substrate 54. The opening of the through hole 52a to the main channel 40 and the opening of the through hole 54a to the main channel 40 form sheath fluid confluence ports 52c and 54c that allow the sheath fluid s to flow into the main channel 40 of the flow path body 12c. It is preferable that the widths of the supply port for the suspension Q and the sheath fluid confluence ports 52c and 54c for the sheath fluid s are substantially the same.

[0050] A sheath liquid junction port 52c is provided on the inner surface 40c of the main flow channel 40, and forms a sheath liquid flow in the main flow channel 40 that contacts the electrodes of the electrode pair 44. The sheath liquid junction port 52c is located downstream of the suspension inlet 55d and upstream of the electrode pair 44. The through-holes 52a and 56a form a sheath liquid flow path 64 in the main flow channel 40, which joins the sheath liquid flow with the suspension Q at a joining section 59. A pipe 21a is connected to the sheath liquid flow path 64. The pipe 21a is connected to the sheath liquid junction port 52c via the first supply port 41a, the through-hole 56a, and the through-hole 52a. A sheath liquid junction port 54c is provided on the inner surface 40c of the main flow channel 40, and forms a sheath liquid flow in the main flow channel 40 that contacts the electrodes of the electrode pair 44. The sheath liquid junction port 54c is disposed downstream of the suspension liquid inlet 55d and upstream of the electrode pair 44. The through-hole 54a and the through-hole 58a form a sheath liquid flow path 65 that joins the sheath liquid flow with the suspension liquid Q at a joining point 59 in the main flow path 40. A pipe 21b is connected to the sheath liquid flow path 65. The pipe 21b is connected to the sheath liquid junction port 54c via the second supply port 41b, the through-hole 58a, and the through-hole 54a.

[0051] In the flow path main body 12c, the flow path substrate 55 may be formed by various known methods.

[0052] In the flow path main body 12c, the suspension Q is supplied from the pipe 21 to the supply port 15, flows into the main flow path 40 from the most upstream portion, and then flows from the main flow path 40 to the discharge flow path 70 and to the discharge port 16. The sheath fluid s is supplied to the first supply port 41a and the second supply port 41b. The sheath fluid s flowing into the first supply port 41a is supplied to the main flow path 40 through the through hole 56a provided in the first introduction part 56 and the through hole 52a provided in the first substrate 52, and then flows from the main flow path 40 to the discharge flow path 70 and to the discharge port 16. The sheath fluid s flowing into the second supply port 41b is supplied to the main flow path 40 through the through hole 58a provided in the second introduction part 58 and the through hole 54a provided in the second substrate 54, and then flows from the main flow path 40 to the discharge flow path 70 and to the discharge port 16. The discharge flow path 70 is formed horizontally, but may be formed obliquely upward.

[0053] In the embodiment shown in FIG. 4 , the sheath liquid flow path is composed of the through-hole 56 a (58 a) and the through-hole 52 a (54 a). However, the sheath liquid flow path may be composed only of the through-hole 52 a (54 a), and the flow path configuration can be appropriately designed depending on the device configuration. Therefore, upstream of the electrode pair 44, as conceptually shown in FIG. 5 , sheath liquid flows composed of the sheath liquid s are formed on both sides of the suspension flow of the suspension Q in opposing directions, forming a three-layer laminated flow of sheath liquid flow / suspension flow / sheath liquid flow, and the three-layer laminated flow flows between the electrode pair 44. In the EP device 10 a, the suspension Q and the sheath liquid s are continuously fed between the electrode pair 44, i.e., into the above-described space 20, in a state in which they form a laminated flow of sheath liquid flow / suspension flow / sheath liquid flow. Then, by applying, for example, a pulsed electric field to the suspension Q in the three-layer laminated flow state by the first electrode 45 and the second electrode 46, the above-described continuous liquid-feed EP process using the sheath liquid flow can be performed. In this case, it is sufficient that the thickness Dm of the suspension flow is 1 to 10 mm at least between the electrode pair 44 and at the upstream end of the electrode pair 44. Note that in Figure 5, the piping 21, piping 21a, 21b, pumps 22a, 22b, 22c, 22d, culture device 23, power supply unit 24, control unit 25, tanks 26, 36, and mixer 30 shown in Figure 4 are not shown.

[0054] As described above, the sheath fluid s supplied from the first supply port 41a flows downstream at an incline and passes through the sheath fluid junction port 52c into the main channel 40. On the other hand, the sheath fluid s supplied from the second supply port 41b flows downstream at an incline and passes through the sheath fluid junction port 54c into the main channel 40. As described above, the thickness of the suspension flow is preferably 1 to 8 mm, more preferably 2 to 5 mm, and even more preferably 2 to 3 mm. Furthermore, by making the total thickness of the sheath fluid flow equal to or less than the thickness of the suspension flow, the dilution rate of the suspension after EP can be kept to 2 times or less.

[0055] Furthermore, although there is no limitation on the thickness ds of the sheath liquid flow (see FIG. 5 ), the thickness of the sheath liquid flow at least between the electrode pair 44 and at the upstream end of the electrode pair 44 is preferably 0.1 mm or more, more preferably 0.2 mm or more, even more preferably 0.3 mm or more, and particularly preferably 0.5 mm or more. By setting the total thickness of the two sheath liquids to be equal to or less than the thickness Dm of the suspension flow, the dilution rate of the suspension after continuous liquid feed EP can be reduced to two times or less. Therefore, it is preferable that the total thickness ds of the two sheath liquid flows (see FIG. 5 ) be equal to or less than the thickness Dm of the suspension flow. The thickness Dm of the suspension flow (see FIG. 5 ) and the thickness ds of the sheath liquid flow (see FIG. 5 ) are adjusted by the flow rates of the suspension Q and the sheath liquid s. More specifically, the thickness of the suspension flow can be measured using a visualization device (not shown) having a shape similar to that of the flow path device. The suspension and water are flowed through the visualization device at predetermined flow rates. The thickness of the suspension flow can be measured because the suspension and water are different colors. The thickness of the suspension flow can be adjusted by adjusting the flow rates of the suspension flow and the sheath liquid flow, based on information on the flow rates of the suspension and water and the thickness of the suspension flow at that time.

[0056] In such a flow path body 12c, the angle formed by the suspension flow and the sheath liquid flow when the suspension flow and the sheath liquid flow are joined, i.e., the angle formed by the main flow path 40 and the through-holes 52a and 54a, is not limited, but is preferably small. ＬThe angle formed by the direction of extension of through hole 52a and the direction of extension of through hole 54a is preferably 90° or less, more preferably 60° or less, even more preferably 45° or less, and even more preferably 30° or less. Furthermore, it is preferable that the difference in flow velocity between the suspension flow and the sheath liquid flow when they are joined together is small. Specifically, the flow velocity of the sheath liquid flow when they are joined together is preferably ±10% or less of the flow velocity of the suspension flow, more preferably ±5% or less, even more preferably ±3% or less, and most preferably ±0%, i.e., the suspension flow and the sheath liquid flow are equal in speed.

[0057] In the flow path body 12c of Figure 4, it is preferable to have O-rings as sealing members (not shown) to prevent leakage of the suspension Q between the first introduction part 56 and the first substrate 52, between the first substrate 52 and the flow path substrate 55, between the flow path substrate 55 and the second substrate 54, and between the second substrate 54 and the second introduction part 58.

[0058] In the flow path device 50 shown in FIG. 4, similarly to the flow path device 11 shown in FIG. 2, a tube 13 is provided in the flow path main body 12c, and at least a portion of the tube 13 faces upward in the vertical direction z. As a result, the EP device 10a shown in FIG. 4 can also obtain the same effect as the EP device 10 shown in FIG. 2. The form in which the tube 13 is provided can be the same as the EP device 10 shown in FIG. 2. For example, as described above, it is preferable that at least the nearby portion 13a of the tube 13 extending from the outlet 16 faces upward in the vertical direction z. Also, for example, it is preferable that a portion of the tube 13 extending 3 cm or more from the outlet 16 faces upward in the vertical direction z from the outlet 16. Also, for example, as shown in FIG. 2, the tube 13 extends horizontally 5 cm from the outlet 16a of the outlet 16, and the tube 13 extends from the outlet 16 to a position P １ At the position P 2 cm or more above the vertical direction z ２ Furthermore, the EP device 10a shown in Fig. 4 may also be configured to have a holding portion 34 (see Fig. 3) that holds the tube 13 at a preset height.

[0059] (Second Example of Electroporation Method) A second example of the electroporation method (EP method) is a method in which a suspension Q containing a biologically derived substance and a bioactive substance and a sheath liquid s are continuously fed between a pair of electrodes 44, i.e., into the above-mentioned space 20, with the suspension Q sandwiched between the sheath liquid s, and an electric field is applied to the suspension Q by the pair of electrodes 44, thereby introducing the bioactive substance into the biologically derived substance. The second example of the EP method uses, for example, the EP device 10a, the culture device 23, the tank 26, and the mixer 30 shown in FIG. 4 . In the second example of the EP method, the culture solution in the culture device 23 is aspirated by the pump 22a of the EP device 10a, and the bioactive substance in the tank 26 is aspirated by the pump 22b, and the culture solution in the culture device 23 and the bioactive substance in the tank 26 are mixed in the mixer 30 to produce suspension Q without replacing the culture medium with an EP buffer. The pump 22a supplies the suspension Q to the main flow path 40 through the pipe 21 and the supply port 15. The pump 22c supplies the sheath fluid s in the tank 36 to the main flow path 40 through the pipe 21a, the first supply port 41a, the through-hole 56a in the first introduction part 56, and the through-hole 52a in the first substrate 52, i.e., through the sheath fluid flow path 64. The pump 22d supplies the sheath fluid s in the tank 36 to the main flow path 40 through the pipe 21b, the second supply port 41b, the through-hole 58a in the second introduction part 58, and the through-hole 54a in the second substrate 54, i.e., through the sheath fluid flow path 65. As a result, a three-layer laminated flow of sheath fluid flow / suspension flow / sheath fluid flow is formed between the electrode pair 44, and the suspension Q and sheath fluid s are continuously transported between the electrode pair 44 in a direction from below to above in the vertical direction z. At this time, the power supply unit 24 applies, for example, a pulse voltage to the electrode pair 44 to perform EP.

[0060] In the second example of the EP device and the second example of the EP method, the liquid feed direction Dd of the suspension Q and the sheath liquid s is also from bottom to top in the vertical direction z. When EP is performed by applying an electric field to the suspension Q using the power supply 24 and bubbles are generated between the opposing first and second electrodes 45 and 46, the generated bubbles have a lower specific gravity than the suspension Q and are therefore more likely to move in the vertical direction z than the suspension Q. Therefore, bubbles generated between the electrode pair 44 are removed from between the electrode pair 44 by moving in the vertical direction z without remaining between the electrode pair 44 due to the flow of the suspension Q and the sheath liquid s, thereby suppressing the adverse effects of the application of an electric field to the suspension Q. This allows the second example of the EP method to properly apply an electric field to the suspension Q, thereby optimizing the introduction efficiency and utilization efficiency of the bioactive substance. Furthermore, even if the composition of the suspension Q is such that bubbles are likely to be generated when an electric field is applied to the suspension Q, if bubbles are generated between the electrode pair 44 as described above, the generated bubbles have a smaller specific gravity than the suspension Q and are therefore more likely to move in the vertical direction z than the suspension Q, and are removed from between the electrode pair 44 by moving in the vertical direction z without remaining between the electrode pair 44 due to the delivery of the suspension Q and sheath fluid s, thereby suppressing the adverse effects of the bubbles caused by the application of an electric field to the suspension Q. As a result, in the second example of the EP method, the electric field can be appropriately applied to the suspension Q, and as a result, the introduction efficiency and the utilization efficiency of the bioactive substance can be optimized.

[0061] Furthermore, in the second example of the EP device and the second example of the EP method, as described above, at least a portion of the tube 13 faces upward in the vertical direction z, making it even more difficult for bubbles generated between the electrode pair 44 and removed from between the electrode pair 44 to be trapped by the outlet 16. This makes it easier for bubbles to be discharged from the flow path main body 12 to the tube 13 along with the flow of the delivered suspension Q, thereby discharging the bubbles to the outside of the flow path main body 12. This allows for proper application of an electric field to the suspension Q and suppresses a decrease in electroporation efficiency. If bubbles are trapped at the outlet 16 and not discharged to the outside of the flow path main body 12c, they may accumulate and invade the main flow path 40. In this case, bubbles that invade the main flow path 40 disrupt the flow velocity distribution of the three-layered flow of sheath liquid flow / suspension flow / sheath liquid flow, thereby reducing the efficiency of introducing a bioactive substance into a biologically derived substance. In the second example of the EP device and the second example of the EP method, the above-described arrangement of the tube 13 also makes it difficult for bubbles to be trapped at the outlet 16, thereby suppressing a decrease in the efficiency of introducing a bioactive substance into the biologically derived material. Furthermore, if trapped bubbles break, the pressure fluctuations caused by the breakage can damage the biologically derived material after EP processing, potentially resulting in a decrease in the quality of the biologically derived material or a decrease in the effectiveness of the introduced bioactive substance. However, in the second example of the EP device and the second example of the EP method, the above-described arrangement of the tube 13 prevents bubbles from being trapped at the outlet 16, thereby suppressing a decrease in the quality of the biologically derived material or a decrease in the effectiveness of the introduced bioactive substance. Furthermore, if there are many trapped bubbles, and the trapped bubbles coalesce and break, the pressure fluctuations caused by the bubbles breaking will be large, causing significant damage to the biological material after EP processing. However, in the second example of the EP device and the second example of the EP method, the above-mentioned arrangement of the tube 13 prevents bubbles from being trapped in the outlet 16, as described above, and therefore also prevents significant damage to the biological material described above.

[0062] In the second example of the EP method, similarly to the first example of the EP method, the above-described culturing step and mixing step are carried out before EP is performed, and the culture medium is not replaced with an EP buffer, i.e., without adding an EP buffer, and the suspension can be mixed with a bioactive substance and continuously fed between the electrode pair 44. In the second example of the EP method, similarly to the first example of the EP method, the culture medium is not replaced with an EP buffer, and therefore, even for suspensions that contain more medium and have higher electrical conductivity than suspensions that are typically subjected to EP, the suspension is subjected to EP while moving vertically from below to above between the electrode pair, and therefore, any bubbles generated between the electrode pair are removed from between the electrode pair by moving vertically, thereby suppressing the adverse effects of the application of an electric field to the suspension caused by the bubbles.

[0063] (Third Example of Electroporation Apparatus) Figure 6 is a schematic cross-sectional view showing a third example of an electroporation apparatus (EP apparatus) according to an embodiment of the present invention. In Figure 6, the same components as those in the EP apparatus 10a shown in Figure 4 are designated by the same reference numerals, and detailed description thereof will be omitted. Compared to the EP apparatus 10a shown in Figure 4, the EP apparatus 10b differs in the configuration of the discharge flow path 71 that connects the main flow path 40 and the discharge port 16, and in the position of the discharge port 16, but otherwise has the same configuration as the EP apparatus 10a shown in Figure 4.

[0064] The discharge flow path 71, like the discharge flow path 70 of the EP device 10a shown in FIG. 4, connects the main flow path 40 and the discharge port 16 and is provided on the second substrate 54. Unlike the discharge flow path 70 of the EP device 10a shown in FIG. 4, the discharge flow path 71 does not have a linear flow path extending parallel to the horizontal plane Hp, but has an inclined first flow path 71a and a linear second flow path 71b extending in the vertical direction z. The first flow path 71a and the second flow path 71b are arranged in succession from the main flow path 40 side. The first flow path 71a is in communication with the other end 40b of the main flow path 40, and the second flow path 71b is in communication with the discharge port 16. The first flow path 71a is a linear flow path that is inclined upward in the vertical direction z from the other end 40b of the main flow path 40. The first flow path 71a is preferably inclined upward in the vertical direction z, which makes it even more difficult for generated bubbles to be trapped in the first flow path 71a and makes it easier for the bubbles to be carried along with the flow of the suspension Q and discharged from the flow path main body 12 to the tube 13. The second flow path 71b is a linear flow path extending in the vertical direction, which means that there are very few places in the second flow path 71b where generated bubbles can be trapped and makes it even easier for the bubbles to be carried along with the flow of the suspension Q and discharged from the flow path main body 12 to the tube 13. For these reasons, the discharge flow path 71 has better bubble discharge performance than the discharge flow path 70 of the EP device 10a shown in FIG.

[0065] As described above, the second flow path 71b communicates with the outlet 16, which is provided on the upper end surface 12d of the flow path main body 12c. The upper end surface 12d of the flow path main body 12c is the end surface on the other end 40b side of the main flow path 40. In the configuration of FIG. 6, the opening surface 16b of the outlet 16 is parallel to the horizontal plane Hp and faces upward. In the configuration of FIG. 6, the angle between the opening surface 16b of the outlet 16 and the horizontal plane Hp is 180°. The tube 13 is connected to the outlet 16. In the configuration of FIG. 6, the tube 13 extends in the vertical direction z and extends straight in the vertical direction z. Note that the tube 13 can be extended in the vertical direction by, for example, configuring the tube 13 to include a holding portion 34 (see FIG. 3) that holds the tube 13 at a predetermined height. In the flow path device 50 shown in Fig. 6, similar to the flow path device 50 shown in Fig. 4, a tube 13 is provided in the flow path main body 12c, and the tube 13 faces upward in the vertical direction z. As a result, the EP device 10b shown in Fig. 6 can also achieve the same effects as the EP device 10a shown in Fig. 4. Furthermore, the discharge flow path 71 is easier to discharge bubbles and has superior bubble discharge performance than the discharge flow path 70 of the EP device 10a shown in Fig. 4. Therefore, the EP device 10b can more appropriately apply an electric field to the suspension than the EP device 10a, and can suppress a decrease in the efficiency of introducing bioactive substances into biologically derived materials.

[0066] In the EP device 10b, the discharge flow path 71 is configured to be provided on the second substrate 54, but this is not limited thereto, and the discharge flow path 71 may be provided on the first substrate 52, for example. The configuration of the above-mentioned discharge flow path 71 is not particularly limited as long as at least a portion of the discharge flow path 71 extends in the vertical direction. In terms of excellent bubble discharge performance, it is preferable that the discharge flow path 71 have a first flow path 71a that is inclined upward in the above-mentioned vertical direction z and a linear second flow path 71b that extends in the vertical direction z.

[0067] (Third Example of Electroporation Method) In the third example of the electroporation method (EP method), similar to the second example of the EP method, the culture solution in the culture device 23 is aspirated by the pump 22a of the EP device 10b, and the bioactive substance in the tank 26 is aspirated by the pump 22b. Without replacing the culture medium with an EP buffer, the culture solution in the culture device 23 and the bioactive substance in the tank 26 are mixed in the mixer 30 to produce the suspension Q. The pump 22a supplies the suspension Q to the main flow path 40 through the piping 21 and the supply port 15. The pump 22c supplies the sheath fluid s in the tank 36 to the main flow path 40 through the piping 21a, the first supply port 41a, the through-hole 56a provided in the first introduction part 56, and the through-hole 52a provided in the first substrate 52, i.e., through the sheath fluid flow path 64. Furthermore, the pump 22d supplies the sheath fluid s in the tank 36 through the piping 21b, the second supply port 41b, the through-hole 58a provided in the second introduction part 58, and the through-hole 54a provided in the second substrate 54, i.e., through the sheath fluid flow path 65, to the main flow path 40. As a result, a three-layer laminated flow of sheath fluid flow / suspension flow / sheath fluid flow is formed between the electrode pair 44, and the suspension Q and sheath fluid s are continuously sent between the electrode pair 44 in a direction from below to above in the vertical direction z. At this time, the power supply unit 24 applies, for example, a pulse voltage to the electrode pair 44 to perform EP.

[0068] In the third example of the EP device 10b and the third example of the EP method, the liquid feed direction Dd of the suspension Q and sheath fluid s is from bottom to top in the vertical direction z. When EP is performed by applying an electric field to the suspension Q using the power supply 24 and bubbles are generated between the opposing first and second electrodes 45 and 46, the generated bubbles have a lower specific gravity than the suspension Q and are therefore more likely to move in the vertical direction z than the suspension Q. Therefore, bubbles generated between the electrode pair 44 are removed from between the electrode pair 44 by moving in the vertical direction z without remaining between the electrode pair 44 due to the flow of the suspension Q and sheath fluid s, thereby suppressing the adverse effects of the application of an electric field to the suspension Q. This allows the application of an electric field to the suspension Q to be properly performed in the third example of the EP method, thereby achieving appropriate introduction efficiency and utilization efficiency of the bioactive substance. Furthermore, even if the composition of the suspension Q is such that bubbles are likely to be generated when an electric field is applied to the suspension Q, if bubbles are generated between the electrode pair 44 as described above, the generated bubbles have a smaller specific gravity than the suspension Q and are therefore more likely to move in the vertical direction z than the suspension Q, and are removed from between the electrode pair 44 by moving in the vertical direction z without remaining between the electrode pair 44 due to the delivery of the suspension Q and sheath fluid s, thereby suppressing the adverse effects of the bubbles caused by the application of an electric field to the suspension Q. As a result, in the third example of the EP method as well, the application of an electric field to the suspension Q can be properly carried out, and as a result, the introduction efficiency and the utilization efficiency of the bioactive substance can be properly maintained.

[0069] Furthermore, in the third example of the EP device 10b and the third example of the EP method, the tube 13 faces upward in the vertical direction z and extends straight, as described above. This makes it even more difficult for bubbles generated between the electrode pair 44 and removed from between the electrode pair 44 to be trapped at the outlet 16. This makes it even more likely for bubbles to be carried along with the flow of the delivered suspension Q and discharged from the flow path main body 12 into the tube 13, allowing the bubbles to be discharged outside the flow path main body 12 more quickly. This allows for even more appropriate application of an electric field to the suspension Q and even more suppression of a decrease in electroporation efficiency. If bubbles are trapped at the outlet 16 and not discharged outside the flow path main body 12c, they may accumulate and invade the main flow path 40. In this case, bubbles that invade the main flow path 40 disrupt the flow velocity distribution of the three-layered flow of sheath liquid flow / suspension flow / sheath liquid flow, thereby reducing the efficiency of introducing a bioactive substance into a biologically derived substance. In the third example of the EP device and the third example of the EP method, the above-described arrangement of the tube 13 further reduces the likelihood of bubbles being trapped at the outlet 16, thereby further suppressing the decrease in the efficiency of introducing a bioactive substance into the biologically derived material. Furthermore, if trapped bubbles break, the resulting pressure fluctuations can damage the biologically derived material after EP treatment, potentially resulting in a decrease in the quality of the biologically derived material or a decrease in the effectiveness of the introduced bioactive substance. However, in the third example of the EP device and the third example of the EP method, the above-described arrangement of the tube 13 further reduces the likelihood of bubbles being trapped at the outlet 16, thereby further suppressing the decrease in the quality of the biologically derived material or the decrease in the effectiveness of the introduced bioactive substance. Furthermore, if there are many trapped bubbles, and the trapped bubbles coalesce and break, the pressure fluctuations caused by the bubbles breaking will be large, causing significant damage to the biological material after EP processing. However, in the third example of the EP device and the third example of the EP method, the above-mentioned arrangement of the tube 13 makes it even more difficult for bubbles to become trapped in the outlet 16, as described above, and therefore further reduces the significant damage to the biological material described above.

[0070] In the third example of the EP method, similarly to the first example of the EP method, the above-described culturing step and mixing step are carried out before EP is performed, and the culture medium is not replaced with an EP buffer, i.e., without adding an EP buffer, and the suspension can be mixed with a bioactive substance and continuously fed between the electrode pair 44. In the third example of the EP method, similarly to the first example of the EP method, the culture medium is not replaced with an EP buffer, and therefore, even for suspensions that contain more medium and have higher electrical conductivity than suspensions that are typically subjected to EP, the suspension is subjected to EP while moving vertically from below to above between the electrode pair, and therefore, any bubbles generated between the electrode pair are removed from between the electrode pair by moving vertically, thereby suppressing the adverse effects of the application of an electric field to the suspension caused by the bubbles.

[0071] In the second and third examples of the EP device and the second and third examples of the EP method described above, continuous liquid flow EP is performed by applying a pulsed electric field to the suspension, for example. However, in practice, continuous liquid flow EP is performed by applying a pulsed electric field to a three-layer laminate flow of sheath liquid flow / suspension flow / sheath liquid flow. Here, to perform proper continuous liquid flow EP, it is necessary to apply a pulsed electric field of an appropriate voltage with an appropriate pulse width (length of one pulse) depending on the concentration of the suspension, etc. The optimal conditions for applying such an electric field are predetermined depending on the steady-state concentration of the suspension, flow rate, and the frequency of the set pulsed electric field, etc. Note that the EP methods described in PCT / JP / 2025 / 016650 or WO 2024 / 248133 may also be used in the first to third examples of the EP method described above.

[0072] The above-described electroporation apparatus and electroporation method can both be used to produce useful substances such as target proteins or viruses. That is, the method for producing useful substances includes the above-described electroporation method. Each part of the EP apparatus will be described below.

[0073] [Distance D Between Electrode Pairs] The distance D between the electrode pair 17 (the distance between the electrode surface 18a of the first electrode 18 and the electrode surface 19a of the second electrode 19) and the distance D between the electrode pair 44 (the distance between the electrode surface 45a of the first electrode 45 and the electrode surface 18a of the second electrode 46) are each preferably 1 to 10 mm. The lower limit of the distance D between the electrode pair is more preferably 2 mm, and even more preferably 3 mm. The upper limit of the distance D between the electrode pair is more preferably 6 mm, and even more preferably 5 mm. If the distance D between the electrode pair is too large, an electric field greater than necessary may be generated, resulting in heat generation, electric field concentration at the electrode ends, boiling or discharge, and a decrease in the survival rate or introduction efficiency of biologically derived materials such as cells. From the viewpoint of preventing heat generation, the distance D between the electrode pair is preferably 1 to 6 mm. Furthermore, a distance D between the electrode pair of 1 to 3 mm is more preferable because it suppresses the generation of heat and discharge and also suppresses a decrease in the survival rate or introduction efficiency of biologically derived materials such as cells due to heat generation or discharge. The distance D between the electrode pair corresponds to the thickness of the flow path substrate 28 (see FIG. 2) and the flow path substrate 55 (see FIG. 4). The distance D between the electrode pair is also referred to as the inter-electrode gap or the gap. The distance D between the electrode pair can be measured by measuring the length of the relevant portion using a vernier caliper or a micrometer.

[0074] <Electrode Pair> The first electrodes 18, 45 and the second electrodes 19, 46 constituting the electrode pair are made of, for example, a metal material or a carbon material. Specifically, the same configuration as the electrodes described in WO 2023 / 157673 can be adopted.

[0075] (Thickness) The thicknesses of the first electrodes 18, 45 and the second electrodes 19, 46 are not particularly limited, but are preferably 0.5 to 10 mm, and more preferably 1 to 5 mm. The thicknesses of the first electrodes 18, 45 and the second electrodes 19, 46 can be measured at the corresponding portions using a vernier caliper or a micrometer.

[0076] <Configuration of Flow Channel Device> The materials constituting the first substrate 27, flow channel substrate 28, and second substrate 29 of the flow channel body 12 in FIG. 2 are not particularly limited. Furthermore, the materials constituting the first introduction section 56, first substrate 52, flow channel substrate 55, second substrate 54, and second introduction section 58 of the flow channel body 12c in FIG. 4 are not particularly limited. Various materials can be used as the materials constituting these components, as long as they have sufficient resistance to the suspension, or the suspension and sheath liquid, and can ensure the necessary rigidity. Note that the EP device may use the device configuration described in PCT / JP / 2025 / 016650 or WO 2024 / 248133, except for the configuration of the tube 13 described above.

[0077] <Suspension> The suspension to be subjected to electroporation (EP) contains a biologically derived substance and a bioactive substance. Furthermore, the suspension does not have the culture medium replaced with an EP buffer, nor does it have an EP buffer added. That is, the suspension does not contain an EP buffer, is not substantially free of salt (e.g., sodium chloride), or is not substantially free of poloxamer. The suspension preferably has an electrical conductivity of 2 mS / cm or higher, more preferably 3 mS / cm or higher, and even more preferably 4 mS / cm or higher. Typically, before EP, the culture medium in the suspension is replaced with an EP buffer, and the medium in the suspension is replaced with an EP buffer. This reduces the electrical conductivity of the suspension, and reduces the generation of bubbles upon application of an electric field. Suspensions with the above electrical conductivities are usually not suitable for EP, but can be used in the method of the present invention because they allow for smooth removal of bubbles. The electrical conductivity (mS / cm) is measured using an electrical conductivity meter on a suspension at 25°C. Electrical conductivity (mS / cm) is synonymous with electrical conductivity, and electrical conductivity has the same units as electrical conductivity. Methods for adjusting electrical conductivity include, for example, adjusting the volume fraction of biologically derived substances contained in the suspension and changing the type of culture medium.

[0078] Typically, the culture medium (liquid components other than biologically derived substances) for the suspension of the EP target contains less than 0.1 g / L of poloxamer because the culture medium is exchanged with an EP buffer. The sodium chloride concentration is also less than 0.01 g / L. On the other hand, in the present invention, the culture medium is not exchanged with an EP buffer, so the poloxamer content of the medium is 0.1 g / L or more. The poloxamer content may be 1 g / L or more, 3 g / L or more, or 5 g / L or more. In the present invention, even if the poloxamer content is high, the adverse effects of the application of an electric field to the suspension Q due to air bubbles can be suppressed. The culture medium for the suspension of the EP target contains sodium chloride, and the sodium chloride content is 0.001 g / L or more, more preferably 0.01 g / L or more, 0.1 g / L or more, or 1 g / L or more. The culture medium for the suspension of the EP target contains inorganic ions, and the total inorganic ion concentration is 10 mmol / L or more, more preferably 50 mmol / L or more, 100 mmol / L or more, or 200 mmol / L or more. The term "inorganic ions" refers to bulk inorganic ions typically added to culture media, including calcium, magnesium, potassium, sodium, chloride, nitrate, phosphate, and sulfate ions. The culture medium for the suspension of the EP target contains sodium ions, and the total sodium ion concentration is 1 mmol / L or more, more preferably 10 mmol / L or more, 20 mmol / L or more, 30 mmol / L or more, 40 mmol / L or more, 50 mmol / L or more, 60 mmol / L or more, 70 mmol / L or more, 80 mmol / L or more, 90 mmol / L or more, or 100 mmol / L or more. In this embodiment, even if the total amount of sodium ions is high, the interference with the application of an electric field to the suspension Q due to bubbles and the decrease in productivity can be suppressed. The content can be determined by centrifuging the suspension at 300 G for 15 minutes, collecting the centrifuged supernatant, and measuring it by high performance liquid chromatography.

[0079] The suspension flow rate is preferably 1 mL / min or more, more preferably 2 mL / min or more, even more preferably 3 mL / min or more, even more preferably 4 mL / min or more, even more preferably 5 mL / min or more, even more preferably 8 mL / min or more, even more preferably 10 mL / min or more, even more preferably 12 mL / min or more, particularly preferably 15 mL / min or more, more particularly preferably 20 mL / min or more, even more particularly preferably 25 mL / min or more, even more particularly preferably 30 mL / min or more, and most preferably 35 mL / min or more. The upper limit of the suspension flow rate is preferably 1000 mL / min. For example, the suspension flow rate is adjusted by a pump that delivers the suspension. A suspension flow rate of 1 mL / min or more is preferable in that it increases the EP throughput. The above flow rate can be measured by installing various flow sensors (ultrasonic, Coriolis, thermal MEMS (Micro Electro Mechanical Systems)) in the delivery flow path. Alternatively, the flow rate may be calculated from the change in weight of the collection container and the liquid transfer time.

[0080] (Biologically derived products) Biologically derived products are not particularly limited, and specific examples include cells, organelles, intracellular granules and vesicles, and bacteria. Among these, cells are preferred, animal cells are more preferred, mammalian cells are even more preferred, and human or Chinese hamster-derived cells are most preferred, due to the superior effects of the present invention. Specific examples of cells include human T cells, HEK (Human Embryonic Kidney) 293, A549, SF9, EB66, Daudi, HeLa, Vero, MDCK, BHK (Baby Hamster Kidney), CHO (Chinese Hamster Ovary), NS0, SP2 / 0, hybridoma, etc. In terms of pharmaceutical production, gene transfer using HEK293 or CHO is most commonly used.

[0081] (Bioactive Substances) Bioactive substances are substances such as nucleic acids (e.g., DNA, RNA) and proteins that, when introduced into a biologically derived substance, exert some effect on the biologically derived substance. Examples of nucleic acids include plasmids, linear DNA, and mRNA (messenger RNA), with plasmids being particularly preferred. The concentration of the bioactive substance in the suspension is preferably 1 to 1000 μg / mL, more preferably 2.5 to 500 μg / mL, and even more preferably 10 to 200 μg / mL. The concentration of the bioactive substance in the suspension relative to the medium in the suspension is preferably 10 to 500 μg / mL. The concentration of the bioactive substance per biologically derived substance in the suspension is preferably 20 pg / unit or less, more preferably 5 pg / unit or less, even more preferably 1 pg / unit or less, and particularly preferably 0.5 pg / unit or less. The lower the concentration of the bioactive substance per biologically derived substance in the suspension, the less the amount of bioactive substance used per biologically derived substance, thereby reducing costs.

[0082] Although there is no limitation on the concentration of the biologically derived substance in suspension Q, it is preferable that the volume fraction of the biologically derived substance in suspension Q is 20% or more. In EP, by increasing the concentration of the biologically derived substance in suspension Q, the amount of biologically derived substance processed can be increased compared to when the same amount of a low-concentration suspension is introduced. For example, doubling the concentration allows for twice the processing, thereby improving the EP throughput and utilization efficiency of the biologically derived substance. In addition, in EP, as described above, bioactive substances are incorporated into the biologically derived substance by electrophoresis. Therefore, even if the concentration of the biologically derived substance is increased, there is no need to increase the amount of bioactive substance accordingly, and the utilization efficiency of the bioactive substance can also be improved.

[0083] By having a volume fraction of biologically derived substances in suspension Q of 20% or more, the above-mentioned effects can be suitably obtained, enabling efficient processing. Furthermore, if the concentration of suspension Q is too high, the viscosity of the cell suspension increases rapidly, potentially clogging the flow path. However, by setting the volume fraction of biologically derived substances in suspension Q to 70% or less, clogging of the flow path caused by an excessively high concentration of suspension Q can be suitably prevented. The volume fraction of biologically derived substances in suspension Q is more preferably 30% or more, more preferably 40% or more, and even more preferably 50% or more. The upper limit of the volume fraction of biologically derived substances in suspension Q is more preferably 65%, and even more preferably 60%. The volume fraction of biologically derived substances in suspension Q can be determined by measuring the average diameter and concentration (number per unit volume) of the biologically derived substances by image analysis (for example, Vi-CELL XR (Beckman Coulter)), and calculating the volume fraction (= 4 ÷ 3 × (average diameter ÷ 2) 3 ×π×concentration×100(%)) can be calculated. The volume fraction is calculated assuming that the biologically derived substances are spherical. More specifically, for example, if the average diameter of the biologically derived substances in suspension Q is 20 μm and the concentration is 40×10 ６ When the volume fraction of the biologically derived material in the suspension Q is 17%, the volume fraction of the biologically derived material in the suspension Q can be calculated to be 17%.

[0084] Liquid media typically used in the culture of animal cells can be used as the culture medium (culture solution). Various companies sell media based on phosphate-buffered saline (PBS) optimized for culturing cells such as HEK293. The medium may contain, but is not limited to, amino acids, salts, sugars (e.g., glucose), vitamins, hormones, growth factors, lipids, trace elements, etc. The pH (hydrogen ion exponent) of the medium is 6 to 8, preferably 6.8 to 7.6, and more preferably 7.2 to 7.6. An antifoaming agent may also be added to the culture medium (culture solution). Silicone-based antifoaming agents are preferred, with dimethicone being particularly preferred. Antifoaming agents containing polydimethylsiloxane are preferred, and polydimethylsiloxane containing finely powdered silica (simethicone) is more preferred. The rate of simethicone addition relative to the volume of culture solution is not particularly limited, but is preferably 5 mg / hr / L or less, more preferably 2 mg / hr / L or less. The medium may contain a block copolymer of polyoxypropylene and polyoxyethylene. As the copolymer of polyoxypropylene and polyoxyethylene, poloxamer is preferred, and poloxamer-188 is more preferred.

[0085] <Sheath Liquid> Various liquids other than a suspension can be used as the sheath liquid flow. A preferred example of a sheath liquid is a culture medium used in culture. As described above, there are two sheath liquid flows. The flow rate of each of the two sheath liquid flows is preferably 0.1 mL / min or more. The upper limit of the flow rate of the sheath liquid is preferably equal to or less than the flow rate of the suspension. For example, the flow rate of the sheath liquid is adjusted by a pump that delivers the sheath liquid. Setting the flow rate of the sheath liquid to 0.1 mL / min or more is preferable in that it can effectively prevent the suspension from contacting the electrodes. Setting the flow rate of the sheath liquid to a value equal to or less than the flow rate of the suspension is also preferable in that it can reduce the amount of sheath liquid and prevent dilution of the suspension.

[0086] Here, in EP, when an electric field is applied, holes open in the membrane (or shell) of the biological material, allowing the bioactive substance to enter the biological material through electrophoresis. The holes formed in the membrane of the biological material gradually close over time, but if the liquid components (osmotic pressure, ion concentration, etc.) outside the biological material suddenly change before the holes close, for example, due to operations such as diluting the suspension, the movement of ions and medium through the holes can cause a sudden change in the component ratio within the biological material, as well as damage such as deformation and expansion. As a result, for example, in the case of substance introduction into cells, the viability of the biological material can decrease, potentially reducing the introduction efficiency and utilization efficiency of the bioactive substance during EP. If the thickness ds of the sheath liquid flow is greater than the thickness Dm of the suspension flow and the dilution rate of the suspension Q is high, for example, in the case of substance introduction into cells, the viability of the biological material can decrease (become damaged), resulting in low EP efficiency. As a result of investigations by the inventors, it was confirmed that, for example, when a cell suspension is diluted with a culture medium sheath fluid flow, a significant decrease in cell viability is often observed when the dilution rate is more than 2. For this reason, as described above, by making the total thickness of the sheath fluid flow at the time of merging with the suspension flow less than the thickness of the suspension flow, i.e., by making the dilution rate of suspension Q less than 2, it is possible to suppress the problems caused by a large amount of sheath fluid s being mixed into the biological material, such as a decrease in the viability of the biological material in the case of substance introduction into cells, and to perform efficient processing.

[0087] In order to more suitably obtain this effect, the total thickness of the sheath liquid flow at the time of merging with the suspension flow is preferably 1 time or less, more preferably 0.8 time or less, and even more preferably 0.5 time or less, of the thickness of the suspension flow, as described above. In other words, the dilution ratio of the suspension Q downstream of the electrode pair is preferably 2 time or less, more preferably 1.8 time or less, and even more preferably 1.5 time or less, as described above.

[0088] <Electroporation Voltage> The electroporation voltage is set so that the electric field applied to the suspension between the electrode pair has a desired electric field strength, taking into account the thickness of the suspension flow, the electrical conductivity (electrical conductivity) of the suspension flow, the thickness of the sheath liquid flow, and the electrical conductivity (electrical conductivity) of the sheath liquid flow. Note that the electrical resistance of the electrodes is preferably sufficiently lower than the resistance of the suspension and sheath liquid, as this may result in a decrease in the introduction efficiency and utilization efficiency of the bioactive substance during EP. The optimal value of the electric field applied to the suspension varies depending on the type and size of the biological material, but is typically approximately 100 to 2000 V / cm. The voltage is preferably a pulse voltage. Furthermore, a bipolar pulse (alternating positive and negative) may be used to uniformly distribute electrode reactions (e.g., gas generation by electrolysis, electrode deterioration, etc.).

[0089] <Pulse Width> The optimum value of the pulse width varies depending on the type of biological substance, etc., but is usually 0.1 to 100 ms (milliseconds), preferably about 1 to 10 ms.

[0090] <Pulse Period (Pulse Interval)> The pulse period is preferably synchronized (an integer multiple of) the time it takes for the biologically derived substance to pass through the electrode (electrode length L (see Figures 1 and 4)). For example, a pulse voltage is applied 1 to 5 times, preferably once, on average while the biologically derived substance passes through the electrode (electrode length L). The time it takes for the biologically derived substance to pass through the electrode (electrode length L) is determined by the flow rate and the cross-sectional area of ​​the flow channel. When the cross-sectional shape of the flow channel is a rectangle with all interior angles at 90°, the cross-sectional area of ​​the flow channel is (electrode width W) × (distance D between the electrode pair). The electrode width W is shown in Figure 1, and the distance D between the electrode pair is shown in Figures 2 and 4.

[0091] This specification incorporates by reference the contents of International Application No. PCT / JP2024 / 012393, including (1) pre-culture of cells, (2) adjustment of cell concentration (concentration, etc.), (3) addition and mixing of nucleic acid, (4) electroporation (gene introduction), (5) culture for virus production (main culture), and (6) virus recovery and purification.

[0092] <Method for producing a useful substance> As described above, the method for producing a useful substance includes the EP method of the present invention. In the method for producing a useful substance, preferably, the biologically derived substance is a cell, and the bioactive substance is a nucleic acid. By using the EP method of the present invention, the nucleic acid is introduced into the cell, and the cell into which the nucleic acid has been introduced is cultured, thereby causing the cell to produce the useful substance.

[0093] The type of useful substance is not particularly limited, but is preferably a protein or a virus. Examples of useful substances include recombinant polypeptide chains, recombinant secreted polypeptide chains, antigen-binding proteins, human antibodies, humanized antibodies, chimeric antibodies, mouse antibodies, bispecific antibodies, Fc fusion proteins, fragmented immune immunoglobulins, single-chain antibodies (scFv), and non-enveloped viruses. More specifically, non-enveloped viruses include adeno-associated viruses, adenoviruses, lentiviruses, baculoviruses, and retroviruses. Non-enveloped viruses are known in the art and are described in International Publication No. WO 2015 / 005430, which is incorporated herein by reference.

[0094] The useful substance is preferably a human antibody, a humanized antibody, a chimeric antibody, a mouse antibody, or an adeno-associated virus. The method for producing the useful substance may include recovery and purification steps.

[0095] The electroporation apparatus and electroporation method of the present invention can be suitably used for the manufacture of gene therapy drugs, etc. The present invention is basically configured as described above. While the electroporation apparatus and electroporation method of the present invention have been described in detail above, the present invention is not limited to the above-described embodiments, and various improvements and modifications may be made without departing from the spirit and scope of the present invention.

[0096] 10, 10a, 10b EP device 11 Flow path device 12 Flow path main body 12a Surface 12b Back surface 12c Flow path main body 13 Tube 13a Nearby portion 14, 40 Main flow path 14a, 14b, 40a, 40b End 14c, 40c Inner surface 15 Supply port 16 Discharge port 16a Outlet 16b Opening surface 17, 44 Electrode pair 18, 45 First electrode 18a, 19a, 45a, 46a Electrode surface 19, 46 Second electrode 20, 47 Space 21, 21a, 21b, 21c, 21d Piping 22a, 22b, 22c, 22d Pump 23 Culture device 24 Power supply unit 25 Control unit 26, 36 Tank 27 First substrate 28 Flow path substrate 28a Opening 29 Second substrate 30 Mixer 32 Connection portion 32a Passage 32b Opening surface 32c Base portion 32d Tapered portion 34 Holding portion 34a Grip portion 34b Support portion 41a First supply port 41b Second supply port 50 Flow path device 52 First substrate 52a, 52b Through holes 52c, 54c Sheath liquid confluence port 54 Second substrate 54a Through hole 55 Flow path substrate 55d Suspension inlet 56 First introduction portion 56a, 56b, 58a Through holes 58 Second introduction portion 59 Confluence portion 64, 65 Sheath liquid flow path 70, 71 Discharge flow path 71a First flow path 71b Second flow path D Distance D Ｌ Extension direction Dd Liquid transfer direction Df Direction Dm Thickness Ds Stacking direction Hp Horizontal plane L Electrode length Q Suspension W Electrode width W １ Distance W ２ Distance ds Thickness s Sheath liquid x, y directions z Vertical direction

Claims

1. An electroporation apparatus including a flow path device used for electroporation, which applies an electric field to a suspension containing a biologically derived substance and a bioactive substance using an electrode pair, thereby introducing the bioactive substance into the biologically derived substance, wherein the flow path device comprises a flow path main body and a tube connected to the flow path main body, the flow path main body has a main flow path extending in the vertical direction, an electrode pair that applies an electric field to the suspension flowing through the main flow path, a supply port that supplies the suspension to the main flow path, and a discharge port that discharges the suspension to which the electric field has been applied to the outside of the flow path device, the tube is connected to the discharge port, and at least a portion of the tube is facing upward in the vertical direction.

2. The electroporation device according to claim 1, wherein the suspension is pumped through the main channel from below to above in the vertical direction.

3. An electroporation apparatus according to claim 1 or 2, wherein at least a portion of the tube extending from the outlet and adjacent thereto faces upward in the vertical direction.

4. An electroporation apparatus according to claim 1 or 2, wherein a portion of the tube extending from the outlet by 3 cm or more faces vertically upward from the outlet.

5. An electroporation apparatus according to claim 1 or 2, wherein the connection portion of the tube at the outlet of the outlet faces upward in the vertical direction.

6. An electroporation apparatus as described in claim 1 or 2, wherein the tube is located at a position extending 5 cm horizontally from the outlet of the outlet and at least 2 cm vertically above.

7. An electroporation device as described in claim 1 or 2, comprising a discharge flow path that connects the main flow path and the discharge port, at least a portion of the discharge flow path extending in the vertical direction, and an outlet of the discharge port facing upward in the vertical direction.

8. An electroporation method comprising the steps of: mixing a culture solution containing a biologically derived substance obtained by culturing with a bioactive substance to obtain a suspension; and using the electroporation device described in claim 1, transporting the suspension vertically from below to above in a main flow path, and applying an electric field to the suspension using an electrode pair to introduce the bioactive substance into the biologically derived substance.

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